There are two major objectives to this three year research project. The first goal is to provide new quantitative and qualitative information concerning the enzymatic process of thyrotropin-releasing hormone (TRH) degradation in four well known sites of TRH action. These are: (1) the peripheral circulation, (2) the Hypophyseal-portal circulation, (3) the pituitary, and (4) the central nervous system. The second objective is to obtain substantive information concerning the physiological and clinical importance of TRH inactivation. Perturbations of the hypothalamic-pituitary-thyroid axis will be the primary paradigm used to evaluate this process. Using solid phase techniques, we will synthesize known or suspected enzymatic fragments of TRH and also incorporate both 3H-proline and 14C-histidine into the TRH molecule. Availability of these peptide markers will enable us to identify and quantitate the rate of appearance of the various TRH split-products by chromatographic methods. Two other techniques will be utilized to quantitate TRH inactivation in vitro. These are: (1) a specific TRH radioimmunoassay, and (2) a sensitive charcoal-absorption assay, developed by the principal investigator. During the course of these studies we will partially purify the plasma TRH deamidase as well as characterize and compare it to other TRH peptidases and a deamidase. Pituitary plasma membranes and CNS synaptosomes will be isolated in order to identify and characterize membrane bound TRH degradases. Present and future research could lead to a basic understanding of how peptide neuroendocrine transmitters are regulated enzymatically, and may permit direct evaluation of thyroid neuroendocrine events in the periphery.